The overall goal of this proposed subproject is to further our understanding of how oral bacteria and their products directly influence the behavior of tissue cells within the periodontium, specifically gingival epithelial cells and connective tissue fibroblasts. The central hypothesis to be tested in this Subproject is that biofilm bacteria exert direct effects on tissue cell behavior within the periodontium. Our specific aims are: 1) to analyze the effects of mediators produced by Actinobacillus actinomycemcomitans (Aa) (released from planktonic or biofilm associated organisms) and microbial-induced mediators (epithelial defensins and supernatants from bacteria stimulated cell cultures) on the migration and proliferation of gingival epithelial cells and fibroblasts; and 2) to determine the effects of Aa-produced mediators and microbial-induced host tissue cell mediators on the synthesis of extracellular matrix proteins, cytokines and other cell signaling molecules by gingival epithelial cells and fibroblasts. Motility of gingival epithelial cells and fibroblasts will be analyzed using time-lapse videomicroscopy and computer-assisted image analysis. Cell migration and proliferation will be studied using an in-vitro wound model, in which a confluent monolayer of epithelial cells and fibroblasts is wounded and repopulation of the created wound space is measured over time. Synthetic activities of cells will be analyzed for production of collagen and non-collagen proteins, prostaglandins, cytokines, defensins, and defensin-like molecules. Although defensins have not been been detected in fibroblasts, this has not been adequately studied in situations where bacteria are in direct contact with connective tissues for extended periods of time. This is relevant to several clinical situations in periodontics, for example, exposed surgical materials, and in certain periodontal diseases characterized by bacterial invasion into gingival connective tissue, such as aggressive periodontitis and necrotizing ulcerative gingivitis. Collectively, this group of studies will further our understanding of the the effects of bacterial biofilms on epithelial and connective tissue cell behavior and metabolism in periodontal diseases and will pave the way for further studies focusing on how these pathogenic mechanisms may become targets for therapeutic intervention.